Enzymatic reactions exhibit remarkable selectivity and efficiency, the likes of which are rarely achieved in bench-top chemical reactions. While it is clear that the biochemical prowess of an enzyme arises from the highly ordered structure of its native-folded protein, the detailed mechanism by which it functions has proven elusive. This is because proteins are not simply static macromolecules that host an active site, as depicted by their crystal structure; rather, they are dynamic molecules whose choreographed motions can gate the transport of substrate to and from the active site and can modulate over time the activity of that site. To develop a mechanistic understanding of how proteins function, it is essential to study this ?choreography of life? at the atomic level with ps time resolution. [unreadable] [unreadable] To probe this choreography of life, we continue to develop and refine the method of picosecond time-resolved X-ray crystallography. This technique exploits the pump-probe method where a laser pulse (pump) triggers a reaction in a protein crystal and a delayed X-ray pulse (probe) takes a ?snapshot? of the protein?s structure. The sequence of time-resolved snapshots can be stitched together into a movie that allows us to literally ?watch? a protein as it functions. These experiments have been conducted on the ID09B time-resolved X-ray beamline at the European Synchrotron and Radiation Facility (ESRF) in Grenoble, France, which is still the only site in the world capable of acquiring time-resolved macromolecular structures with 150-ps time resolution and < 2-? spatial resolution. Unfortunately, the ESRF operates in a mode that is optimized for these studies only 2 weeks out of each year, and this limited amount of beam time has hindered progress in this field. To expand significantly the amount of beam time available for our studies, we have partnered with the Advanced Photon Source (APS) in Argonne, IL to develop picosecond time-resolved X-ray capabilities on BioCARS, an NIH-funded beamline that is operated by the University of Chicago. Orders for the capital equipment required to develop this capability have been placed, and installation of this equipment will take place in FY 2007. When completed, the BioCARS beamline will provide state-of-the-art capabilities in picosecond time-resolved crystallography, and will boost significantly the amount of beam time available to pursue such studies. [unreadable] [unreadable] Time-resolved Laue crystallography requires that the protein be crystallized, and the intermolecular forces that maintain crystalline order can constrain large amplitude conformational motion. This loss of conformational flexibility may perturb, and can even inhibit the function of a protein. As discussed in [Anfinrud and Schotte, Science (2005)], X-rays can extract structural information from molecules in solution, where the full range of conformational motion is permitted. However, that information is not at atomic resolution. We have been working to extend this approach by developing the technique of picosecond time-resolved small angle and wide angle X-ray scattering (SAXS/WAXS), and aim to use this method to probe large amplitude motion in proteins. Preliminary investigations using photolyzed carbon moxymyoglobin (MbCO) as a model system show that this technique is sensitive to the subtle tertiary conformational changes that occur when the protein evolves from its carboxy to its deoxy form. This result is quite encouraging, and has spurred us to continue our efforts to develop this new experimental methodology. [unreadable] [unreadable] In our studies of ligand migration in Mb, we use a laser pulse to trigger dissociation of CO from the heme binding site, and then use an X-ray pulse to probe the time-resolved structure of the protein. We identified numerous sites in which CO becomes transiently trapped, and observed correlated motion of the protein side chains. These observations are based on electron density maps, which provide a visual, but qualitative view of the structural changes. To quantify the time-dependent population and the time-dependent displacement of the atomic coordinates, detailed modeling of our data is required. In collaboration with the group of Prof. George Phillips at the Univ. of Wisconsin, we employed a difference-refinement approach to generate atomic models that were constrained by our picosecond time-resolved electron density maps [Aranda et al., Acta Crystallogr D Biol Crystallogr (2006)]. This study revealed a nonlinear progression of the structure toward the final, deoxy state, and demonstrated that the data quality is sufficient to define atomic coordinates with high precision. In collaboration with the group of Prof. Brunori from the Univ. of Rome, we studied a novel triple mutant of myoglobin, known as YQR Mb [Bourgeois, et al. PNAS (2006)]. We focused on the early time dynamics and assessed the structural origins for the non-exponential, distributed kinetics observed as the protein structure evolved from its carboxy to its deoxy state. [unreadable] [unreadable] Our collaboration with the group of Dr. Gerhard Hummer is currently focusing on small angle scattering of proteins. Preliminary results indicate that computational methods are capable of reproducing fine details observed in time-resolved SAXS/WAXS studies of photolyzed MbCO. Our collaboration with Dr. Eric Henry to develop improved methods for analyzing Laue data has led to a new, automated method for determining the orientation of the protein crystal, a necessary first step in what has, in the past, been a tedious process. We are expanding our studies to more protein systems, and with our combination of time-resolved spectroscopic, crystallographic, and computational tools, we will explore functionally-important structure transitions at an unprecedented level of detail, from which a far more meaningful mechanistic description of protein function will be achieved.